The Lysine Acetyltransferase PCAF Functionally Interacts with Estrogen Receptor Alpha in the Hippocampus of Gonadally Intact Male-But Not Female-Rats to Enhance Short-Term Memory.

Acetylation of histone proteins by histone acetyltransferases (HATs), and the resultant change in gene expression, is a well-established mechanism necessary for long-term memory (LTM) consolidation, which is not required for short-term memory (STM). However, we previously demonstrated that the HAT p300/CBP-associated factor (PCAF) also influences hippocampus (HPC)-dependent STM in male rats. In addition to their epigenetic activity, HATs acetylate nonhistone proteins involved in nongenomic cellular processes, such as estrogen receptors (ERs). Given that ERs have rapid, nongenomic effects on HPC-dependent STM, we investigated the potential interaction between ERs and PCAF for STM mediated by the dorsal hippocampus (dHPC). Using a series of pharmacological agents administered directly into the dHPC, we reveal a functional interaction between PCAF and ERα in the facilitation of short-term object-in-place memory in male but not female rats. This interaction was specific to ERα, while ERß agonism did not enhance STM. It was further specific to dHPC STM, as the effect was not present in the dHPC for LTM or in the perirhinal cortex. Further, while STM required local (i.e., dHPC) estrogen synthesis, the facilitatory interaction effect appeared independent of estrogens. Finally, western blot analyses demonstrated that PCAF activation in the dHPC rapidly (5 min) activated downstream estrogen-related cell signaling kinases (c-Jun N-terminal kinase and extracellular signal-related kinase). Collectively, these findings indicate that PCAF, which is typically implicated in LTM through epigenetic processes, also influences STM in the dHPC, possibly via nongenomic ER activity. Critically, this novel PCAF-ER interaction might exist as a male-specific mechanism supporting STM.

Age-dependent attenuation of spatial memory deficits by the histone acetyltransferase p300/CBP-associated factor (PCAF) in 3xTG Alzheimer's disease mice.

Histone acetylation, catalyzed by histone acetyltransferases, has emerged as a promising therapeutic strategy in Alzheimer's disease (AD). By longitudinally characterizing spatial memory at 3, 6, and 9 mo of age, we show that acute activation and inhibition of the histone acetyltransferase PCAF remediated memory impairments in 3xTG-AD mice in an age-related bidirectional manner. At 3 and 6 mo of age, PCAF activation ameliorated memory deficits. At 9 mo of age, PCAF activation had no effect on spatial memory, whereas PCAF inhibition improved memory deficits in females. This work reveals a complex potential therapeutic role for PCAF in AD, initially benefitting memory but becoming detrimental as the disease progresses.

Reverse transcriptase inhibition potentiates target therapy in BRAF-mutant melanomas: effects on cell proliferation, apoptosis, DNA-damage, ROS induction and mitochondrial membrane depolarization.

Target therapies based on BRAF and MEK inhibitors (MAPKi) have changed the therapeutic landscape for metastatic melanoma patients bearing mutations in the BRAF kinase. However, the emergence of drug resistance imposes the necessity to conceive novel therapeutic strategies capable to achieve a more durable disease control. In the last years, retrotransposons laying in human genome have been shown to undergo activation during tumorigenesis, where they contribute to genomic instability. Their activation can be efficiently controlled with reverse transcriptase inhibitors (RTIs) frequently used in the treatment of AIDS. These drugs have demonstrated anti-proliferative effects in several cancer models, including also metastatic melanoma. However, to our knowledge no previous study investigated the capability of RTIs to mitigate drug resistance to target therapy in BRAF-mutant melanomas. In this short report we show that the non-nucleoside RTI, SPV122 in combination with MAPKi strongly inhibits BRAF-mutant melanoma cell growth, induces apoptosis, and delays the emergence of resistance to target therapy in vitro. Mechanistically, this combination strongly induces DNA double-strand breaks, mitochondrial membrane depolarization and increased ROS levels. Our results shed further light on the molecular activity of RTI in melanoma and pave the way to their use as a novel therapeutic option to improve the efficacy of target therapy. Video Abstract.

Novel 2-substituted-benzimidazole-6-sulfonamides as carbonic anhydrase inhibitors: synthesis, biological evaluation against isoforms I, II, IX and XII and molecular docking studies.

Inhibition of Carbonic Anhydrases (CAs) has been clinically exploited for many decades for a variety of therapeutic applications. Within a research project aimed at developing novel classes of CA inhibitors (CAIs) with a proper selectivity for certain isoforms, a series of derivatives featuring the 2-substituted-benzimidazole-6-sulfonamide scaffold, conceived as frozen analogs of Schiff bases and secondary amines previously reported in the literature as CAIs, were investigated. Enzyme inhibition assays on physiologically relevant human CA I, II, IX and XII isoforms revealed a number of potent CAIs, showing promising selectivity profiles towards the transmembrane tumor-associated CA IX and XII enzymes. Computational studies were attained to clarify the structural determinants behind the activities and selectivity profiles of the novel inhibitors.

Identification of Limonol Derivatives as Heat Shock Protein†90 (Hsp90) Inhibitors through a Multidisciplinary Approach.

The identification of inhibitors of Hsp90 is currently a primary goal in the development of more effective drugs for the treatment of various types of multidrug resistant malignancies. In an attempt to identify new small molecules modulating the activity of Hsp90, we screened a small library of tetranortriterpenes. A high-affinity interaction with Hsp90 inducible form was uncovered for eight of these compounds, five of which are described here for the first time. By monitoring the ATPase activity and the citrate synthase thermal induced aggregation, compound 1 (cedrelosin A), 3 (7α-limonylacetate), and 5 (cedrelosin B), containing a limonol moiety, were found to be the most effective in compromising the Hsp90α chaperone activity. Consistent with these findings, the three compounds caused a depletion of c-Raf and pAkt Hsp90 client proteins in HeLa and MCF/7 cell lines. Induced fit docking protocol and molecular dynamics were used to rationalize the structural basis of the biological activity of the limonol derivatives. Taken together, these results point to limonol-derivatives as promising scaffolds for the design of novel Hsp90α inhibitors.

Successes and challenges in the development of BD1-selective BET inhibitors: a patent review.

INTRODUCTION: Bromodomain and ExtraTerminal (BET) domain proteins are transcriptional cofactors that, recognizing acetylated lysines of histone and non-histone proteins, can modulate gene expression. The BET family consists of four members, each of which contains two bromodomains (BD1 and BD2) able to recognize the acetylated mark. Pan-BET inhibitors (BETi) have shown a promising anticancer potential in many clinical trials; however, their further development has been in part hampered by the side effects due to their lack of selectivity. Mounting evidence suggests that BD1 is primarily involved in cancer and that its selective inhibition can phenocopy the anticancer effects of pan-BETi with increased tolerability. Therefore, the development of BD1 selective inhibitors is highly pursed in both academia and industry. AREAS COVERED: This review aims at giving an overview of the patent literature of BD1-selective BETi between 2014 and 2023. WIPO, USPTO, EPO, and SciFinder® databases were used for the search of patents. EXPERT OPINION: The development of BD1-selective BETi, despite challenging, is highly desirable as it could have a great impact on the development of new safer anticancer therapeutics. Several strategies could be applied to discover potent and selective compounds with limited side effects.

Prodrug Approach to Exploit (S)-Alanine Amide as Arginine Mimic Moiety in the Development of Protein Arginine Methyltransferase 4 Inhibitors.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as an emerging target class in oncology and other diseases. A successful strategy to identify PRMT substrate-competitive inhibitors has been to exploit chemical scaffolds able to mimic the arginine substrate. (S)-Alanine amide moiety is a valuable arginine mimic for the development of potent and selective PRMT4 inhibitors; however, its high hydrophilicity led to derivatives with poor cellular outcomes. Here, we describe the development of PRMT4 inhibitors featuring a central pyrrole core and an alanine amide moiety. Rounds of optimization, aimed to increase lipophilicity and simultaneously preserve the inhibitory activity, produced derivatives that, despite good potency and physicochemical properties, did not achieve on-target effects in cells. On the other hand, masking the amino group with a NAD(P)H:quinone oxidoreductase 1 (NQO1)-responsive trigger group, led to prodrugs able to reduce arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155). These results indicate that prodrug strategies can be successfully applied to alanine-amide containing PRMT4 inhibitors and provide an option to enable such compounds to achieve sufficiently high exposures in vivo.

p300/CBP-associated factor selectively regulates the extinction of conditioned fear.

It is well established that the activity of chromatin-modifying enzymes is crucial for regulating gene expression associated with hippocampal-dependent memories. However, very little is known about how these epigenetic mechanisms influence the formation of cortically dependent memory, particularly when there is competition between opposing memory traces, such as that which occurs during the acquisition and extinction of conditioned fear. Here we demonstrate, in C57BL/6 mice, that the activity of p300/CBP-associated factor (PCAF) within the infralimbic prefrontal cortex is required for long-term potentiation and is necessary for the formation of memory associated with fear extinction, but not for fear acquisition. Further, systemic administration of the PCAF activator SPV106 enhances memory for fear extinction and prevents fear renewal. The selective influence of PCAF on fear extinction is mediated, in part, by a transient recruitment of the repressive transcription factor ATF4 to the promoter of the immediate early gene zif268, which competitively inhibits its expression. Thus, within the context of fear extinction, PCAF functions as a transcriptional coactivator, which may facilitate the formation of memory for fear extinction by interfering with reconsolidation of the original memory trace.

Antifungal activity of azole compounds CPA18 and CPA109 against azole-susceptible and -resistant strains of Candida albicans.

OBJECTIVES: In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains. METHODS: Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity. RESULTS: Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition. CONCLUSIONS: Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

Synthesis and cytotoxic activity evaluation of 2,3-thiazolidin-4-one derivatives on human breast cancer cell lines.

It is well known that resveratrol (RSV) displayed cancer-preventing and anticancer properties but its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. Aim of this work was to synthesize pharmacologically active resveratrol analogs with an enhanced structural rigidity and bioavailability. In particular, we have synthesized a library of 2,3-thiazolidin-4-one derivatives in which a thiazolidinone nucleus connects two aromatic rings. Some of these compounds showed strong inhibitory effects on breast cancer cell growth. Our results indicate that some of thiazolidin-based resveratrol derivatives may become a new potent alternative tool for the treatment of human breast cancer.

NADPH Oxidases: From Molecular Mechanisms to Current Inhibitors.

NADPH oxidases (NOXs) form a family of electron-transporting membrane enzymes whose main function is reactive oxygen species (ROS) generation. Strong evidence suggests that ROS produced by NOX enzymes are major contributors to oxidative damage under pathologic conditions. Therefore, blocking the undesirable actions of these enzymes is a therapeutic strategy for treating various pathological disorders, such as cardiovascular diseases, inflammation, and cancer. To date, identification of selective NOX inhibitors is quite challenging, precluding a pharmacologic demonstration of NOX as therapeutic targets in vivo. The aim of this Perspective is to furnish an updated outlook about the small-molecule NOX inhibitors described over the last two decades. Structures, activities, and in vitro/in vivo specificity are discussed, as well as the main biological assays used.

Identification of a Protein Arginine Methyltransferase 7 (PRMT7)/Protein Arginine Methyltransferase 9 (PRMT9) Inhibitor.

Less studied than the other protein arginine methyltransferase isoforms, PRMT7 and PRMT9 have recently been identified as important therapeutic targets. Yet, most of their biological roles and functions are still to be defined, as well as the structural requirements that could drive the identification of selective modulators of their activity. We recently described the structural requirements that led to the identification of potent and selective PRMT4 inhibitors spanning both the substrate and the cosubstrate pockets. The reanalysis of the data suggested a PRMT7 preferential binding for shorter derivatives and prompted us to extend these structural studies to PRMT9. Here, we report the identification of the first potent PRMT7/9 inhibitor and its binding mode to the two PRMT enzymes. Label-free quantification mass spectrometry confirmed significant inhibition of PRMT activity in cells. We also report the setup of an effective AlphaLISA assay to screen small molecule inhibitors of PRMT9.

Lysine methyltransferase inhibitors: where we are now.

Protein lysine methyltransferases constitute a large family of epigenetic writers that catalyse the transfer of a methyl group from the cofactor S-adenosyl-l-methionine to histone- and non-histone-specific substrates. Alterations in the expression and activity of these proteins have been linked to the genesis and progress of several diseases, including cancer, neurological disorders, and growing defects, hence they represent interesting targets for new therapeutic approaches. Over the past two decades, the identification of modulators of lysine methyltransferases has increased tremendously, clarifying the role of these proteins in different physio-pathological states. The aim of this review is to furnish an updated outlook about the protein lysine methyltransferases disclosed modulators, reporting their potency, their mechanism of action and their eventual use in clinical and preclinical studies.

Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction-Reconstruction and Fragment-Growing Approach.

Protein arginine methyltransferases (PRMTs) are important therapeutic targets, playing a crucial role in the regulation of many cellular processes and being linked to many diseases. Yet, there is still much to be understood regarding their functions and the biological pathways in which they are involved, as well as on the structural requirements that could drive the development of selective modulators of PRMT activity. Here we report a deconstruction-reconstruction approach that, starting from a series of type I PRMT inhibitors previously identified by us, allowed for the identification of potent and selective inhibitors of PRMT4, which regardless of the low cell permeability show an evident reduction of arginine methylation levels in MCF7 cells and a marked reduction of proliferation. We also report crystal structures with various PRMTs supporting the observed specificity and selectivity.

Discovery of Benzo[d]imidazole-6-sulfonamides as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with Selectivity for the First Bromodomain.

The bromodomain and extra-terminal (BET) family of proteins includes BRD2, BRD3, BRD4, and the testis-specific protein, BRDT, each containing two N-terminal tandem bromodomain (BRD) modules. Potent and selective inhibitors targeting the two bromodomains are required to elucidate their biological role(s), with potential clinical applications. In this study, we designed and synthesized a series of benzimidazole-6-sulfonamides starting from the azobenzene compounds MS436 (7 a) and MS611 (7 b) that exhibited preference for the first (BD1) over the second (BD2) BRD of BET family members. The most-promising compound (9 a) showed good binding potency and improved metabolic stability and selectivity towards BD1 with respect to the parent compounds.

Modulation of the activity of histone acetyltransferases by long chain alkylidenemalonates (LoCAMs).

A novel class of KAT modulators (long chain alkylidenemalonates, LoCAMs) has been identified. Variations of the alkyl chain length can change the activity profile from inhibition of both KAT3A/KAT2B (as derivative 2a) to the peculiar profile of pentadecylidenemalonate 1b, the first activator/inhibitor of histone acetyltransferases. Together with the powerful apoptotic effect (particularly notable if considering that anacardic acid and other KAT inhibitors are not cell permeable) appoint them as valuable biological tools to understand the mechanisms of lysine acetyltransferases.

Patent evaluation of WO2019209182 (A1) 2019-10-31 (Conjugated Oligoelectrolytes as Antimicrobial Agents).

INTRODUCTION: The insurgence of antibiotic resistance represents one of the biggest public health challenges of our times. During the years, different compounds were developed to fight against resistant bacterial cells, exploiting different mechanisms of action. AREAS COVERED: The patent application describes a set of antimicrobial compounds bearing to the class of the conjugated oligoelectrolytes (COEs). These are molecules characterized by hydrophobic conjugated backbone and terminal polar ionic pendants, able to intercalate into lipid bilayers of bacterial cells. The patent reports the preparation of 15 new compounds and the evaluation of their antimicrobial effect against ESKAPE pathogens ( E nterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.). EXPERT OPINION: The preparation of the compounds claimed is simple and the preliminary activity data are very interesting. Among the claimed compounds, COE-D8, COE-T42, and COE-T62 have the ability to strongly inhibit the bacterial growth at doses similar to the ones of last resource antibiotics. Unfortunately, no in-vivo data are reported. Moreover, the presence of several quaternary amines limits the potential application of these compounds only to topical uses.

Reverse transcriptase inhibitors promote the remodelling of nuclear architecture and induce autophagy in prostate cancer cells.

Emerging data indicate that the reverse transcriptase (RT) protein encoded by LINE-1 transposable elements is a promising cancer target. Nonnucleoside RT inhibitors, e.g. efavirenz (EFV) and SPV122.2, reduce proliferation and promote differentiation of cancer cells, concomitant with a global reprogramming of the transcription profile. Both inhibitors have therapeutic anticancer efficacy in animal models. Here we have sought to clarify the mechanisms of RT inhibitors in cancer cells. We report that exposure of PC3 metastatic prostate carcinoma cells to both RT inhibitors results in decreased proliferation, and concomitantly induces genome damage. This is associated with rearrangements of the nuclear architecture, particularly at peripheral chromatin, disruption of the nuclear lamina, and budding of micronuclei. These changes are reversible upon discontinuation of the RT-inhibitory treatment, with reconsititution of the lamina and resumption of the cancer cell original features. The use of pharmacological autophagy inhibitors proves that autophagy is largely responsible for the antiproliferative effect of RT inhibitors. These alterations are not induced in non-cancer cell lines exposed to RT inhibitors. These data provide novel insight in the molecular pathways targeted by RT inhibitors in cancer cells.

Discovery of a Novel Chemotype of Histone Lysine Methyltransferase EHMT1/2 (GLP/G9a) Inhibitors: Rational Design, Synthesis, Biological Evaluation, and Co-crystal Structure.

Since the discovery of compound BIX01294 over 10 years ago, only a very limited number of nonquinazoline inhibitors of H3K9-specific methyltransferases G9a and G9a-like protein (GLP) have been reported. Herein, we report the identification of a novel chemotype for G9a/GLP inhibitors, based on the underinvestigated 2-alkyl-5-amino- and 2-aryl-5-amino-substituted 3 H-benzo[ e][1,4]diazepine scaffold. Our research efforts resulted in the identification 12a (EML741), which not only maintained the high in vitro and cellular potency of its quinazoline counterpart, but also displayed improved inhibitory potency against DNA methyltransferase 1, improved selectivity against other methyltransferases, low cell toxicity, and improved apparent permeability values in both parallel artificial membrane permeability assay (PAMPA) and blood-brain barrier-specific PAMPA, and therefore might potentially be a better candidate for animal studies. Finally, the co-crystal structure of GLP in complex with 12a provides the basis for the further development of benzodiazepine-based G9a/GLP inhibitors.

Benzothiopyranoindole- and pyridothiopyranoindole-based antiproliferative agents targeting topoisomerases.

New benzothiopyranoindoles (5a-l) and pyridothiopyranoindoles (5m-t), featuring different combinations of substituents (H, Cl, OCH3) at R2-R4 positions and protonatable R1-dialkylaminoalkyl chains, were synthesized and biologically assayed on three human tumor cell lines, showing significant antiproliferative activity (GI50 values spanning from 0.31 to 6.93 µM) and pro-apoptotic effect. Linear flow dichroism experiments indicate the ability of both chromophores to form a molecular complex with DNA, following an intercalative mode of binding. All compounds displayed a moderate ability to inhibit the relaxation activity of both topoisomerases I and II, reasonably correlated to their intercalative capacities. Cleavable assay performed with topoisomerase I revealed a significant poisoning effect for compounds 5g, 5h, 5s, and 5t. A theoretical model provided by hydrated docking calculations clarified the role of the R1-R4 substituents on the topoisomerase I poison activity, revealing a crucial role of the R2-OCH3 group.